This proposal describes a ligand and structure-based approach to discovery and development of low molecular weight gp41 inhibitors effective against Human Immunodeficiency Virus (HIV-1) fusion. Fusion inhibitors possess excellent characteristics for interrupting HIV transmission and preventing disease progression, since they block initial infection of healthy cells and cell-to-cell spread of infection. Yet there are currenly no highly potent small molecule inhibitors of fusion, and peptide fusion inhibitors possess some undesirable characteristics such as high cost and susceptibility to proteolysis. Our central hypothesis is that potent small molecule fusion inhibitors targeting a known hydrophobic pocket can be rationally designed by understanding the molecular features contributing to potency and evaluating the binding mode. The objective of this application is to study and modify promising small molecules to find conditions for determining their structure bound to gp41. We have developed hydrophobic pocket binding indole compounds with IC50's down to 200nM for inhibition of virus - cell and cell - cell fusion. We have also observed that excess negative charge has a deleterious effect on the inhibitors, which we attribute to the effect of the adjacent plasma membrane. We have developed novel reverse hairpin proteins (RH's) as suitable receptors to use in protein - ligand structure studies. They present exposed hydrophobic pocket binding grooves in solution. The goals of the application are to modify the non-peptide fusion inhibitors to assess the determinants of potency, and to identify conditions for studying high resolution structural details of their complexes with gp41. We will accomplish our goals in three Specific Aims: (1) Perform ligand-based design on indole inhibitors, using SAR to predict reliable binding poses; (2) Determine conditions for crystallizing indole inhibitors with RH's to obtain high resolution structures of the complexes; (3) Determine conditions for studying RH - ligand complexes by NMR, using carrier proteins to ensure solubility of the ligands if necessary, and incorporating 19F nuclei as sensitive probes of the environment and interactions. The combination of these experiments will yield an improved understanding of the interaction between small molecule inhibitors and gp41, and the methodology to study them in detail. The significance of this proposal lies in its potential to contribute specifically to HIV-1 fusion inhiitor development and also to the development of techniques and resources applicable to other Class 1 viral fusion proteins, a broader goal relevant to NIH's mission of enhancing fundamental knowledge to treat human disease. The long-term goal of the project is to generate drug-like compounds active against HIV-1 fusion, suitable for therapeutic or microbicide use, which could be used to counter or prevent the millions of new infections that occur annually worldwide. Such compounds could have the added benefit of suppressing viral rebound and overcoming the virus' defenses against neutralizing antibodies.